The eye develops from three cell populations, ectoderm, neuroepithelium, and neural crest cells. A great deal is known about the early stages of eye development, the differentiation of the lens and the formation of the neural retina. However, the differentiation of the anterior part of the optic cup into the ciliary body and the iris structures remains obscure. The long-term goal of investigator is to determine the transcription factors that control the specification of the ciliary epithelium at the anterior of the optic cup and that regulate the development of this structure. To achieve this goal transcriptional mechanism controlling the long-isoform of collagen alpha1 (IX) gene expression in the ciliary epithelium will be investigated. This gene is expressed only in the prospective ciliary epithelium in the early stages of development. Therefore, knowing its transcriptional regulation will provide information on the regulation of ciliary epithelium differentiation. In preliminary studies, the proximal promoter of the collagen alpha1 (IX) gene was analyzed. Two fragments that bind transcription factors were identified. Two proteins, cZic2 and cDtx2 that may bind to the proximal promoter were identified in a one-hybrid screen. To identify additional cis-elements, this application will use a new approach, which permits one to study the regulation of gene expression in the living embryo. Vectors carrying different promoter fragments fused with reporter gene, GFP, will be introduced into the developing eye by in ovo microelectroporation. In preliminary studies, a fragment of the collagen alpha1(IX) gene was identified using this method that is sufficient to drive the expression of a reporter gene in the differentiating ciliary epithelium. The Specific Aims of this application are to (1) identify the cis-elements in the collagen alpha1(IX) promoter that are essential for transcriptional regulation in the ciliary epithelium, (2) analyze the binding specificity of cZic2 and cDtx2 proteins to the proximal promoter of the collagen alphal(IX) gene in vitro and in vivo, and (3) identify the transcription factors that bind to the ciliary epithelium-specific cis-acting elements (identified in the first specific aim) using a one-hybrid screen. Finally, mouse and human homologs of the ciliary epithelium-specific transcription factors will be obtained by searching DNA and protein databases and their relevance to human hereditary eye diseases will be evaluated.